Time filter

Source Type

Rodriguez-Casuriaga R.,Institute Investigaciones Biologicas Clemente Estable IIBCE | Geisinger A.,Institute Investigaciones Biologicas Clemente Estable IIBCE | Santinaque F.F.,Servicio de Citometria de Flujo y Clasificacion Celular SECIF | Lopez-Carro B.,Servicio de Citometria de Flujo y Clasificacion Celular SECIF | Folle G.A.,Servicio de Citometria de Flujo y Clasificacion Celular SECIF
Cytometry Part A | Year: 2011

Mammalian spermatogenesis is still nowadays poorly understood at the molecular level. Testis cellular heterogeneity is a major drawback for spermatogenic gene expression studies, especially when research is focused on stages that are usually very short and poorly represented at the cellular level such as initial meiotic prophase I (i.e., leptotene [L] and zygotene [Z]). Presumably, genes whose products are involved in critical meiotic events such as alignment, pairing and recombination of homologous chromosomes are expressed during the short stages of early meiotic prophase. Aiming to characterize mammalian early meiotic gene expression, we have found the guinea pig (Cavia porcellus) as an especially attractive model. A detailed analysis of its first spermatogenic wave by flow cytometry (FCM) and optical microscopy showed that guinea pig testes exhibit a higher representation of early meiotic stages compared to other studied rodents, partly because of their longer span, and also as a result of the increased number of cells entering meiosis. Moreover, we have found that adult guinea pig testes exhibit a peculiar 4C DNA content profile, with a bimodal peak for L/Z and P spermatocytes that is absent in other rodents. Besides, we show that this unusual 4C peak allows the separation by FCM of highly pure L/Z spermatocyte populations aside from pachytene ones, even from adult individuals. To our knowledge, this is the first report on an accurate and suitable method for highly pure early meiotic prophase cell isolation from adult mammals, and thus sets an interesting approach for gene expression studies aiming at a deeper understanding of the molecular groundwork underlying male gamete production. © 2011 International Society for Advancement of Cytometry © 2011 International Society for Advancement of Cytometry.


Rodriguez-Casuriaga R.,Institute Investigaciones Biologicas Clemente Estable IIBCE | Santinaque F.F.,Servicio de Citometria de Flujo Y Clasificacion Celular SECIF | Folle G.A.,Servicio de Citometria de Flujo Y Clasificacion Celular SECIF | Souza E.,Institute Investigaciones Biologicas Clemente Estable IIBCE | And 3 more authors.
MethodsX | Year: 2014

(Figure Presented). Availability of purified or highly enriched fractions representing the various spermatogenic stages is a usual requirement to study mammalian spermatogenesis at the molecular level. Fast preparation of high quality testicular cell suspensions is crucial when flow cytometry (FCM) is chosen to accomplish the stage/s purification. Formerly, we reported a method to rapidly obtain good quality rodent testicular cell suspensions for FCM analysis and sorting. Using that method we could distinguish and purify early meiocytes (leptotene/ zygotene stages, L/Z) from more advanced ones (pachytene, P) in guinea pig, which presents an unusually high content of early stages. Here we present an upgrade of that method with improvements that enabled the obtainment of high-purity meiotic substages also from mouse testis, namely: • Shortening of the mechanical disaggregation time to optimize the integrity of the suspension. • Elimination of the 25 μm-filtration step to ensure the presence of large P cells. • Inclusion of a non-cytotoxic, DNA-specific, 488 nm-excitable vital fluorochrome (Vybrant DyeCycle Green [VDG], Invitrogen) instead of Hoechst 33342 (requires UV laser, which can damage nucleic acids) or propidium iodide (usually related to dead/damaged cells). As far as we know, this is the first report on the use of this fluorochrome for the discrimination and purification of meiotic prophase I substages. © 2014 The Authors. Published by Elsevier B.V.

Loading Servicio de Citometria de Flujo y Clasificacion Celular SECIF collaborators
Loading Servicio de Citometria de Flujo y Clasificacion Celular SECIF collaborators